We are interested in the processes by which certain red cell proteins are totally lost during differentiation while others, necessary for mature red cell metabolism are spared. Initially, we will focus on pyruvate kinase (PK), an enzyme that is known to undergo a shift in isozyme pattern as well as a 25-fold decrease in total activity during erythroid development. By what processes is elimination of PK from the cell restricted in order to maintain a concentration critical for red cell glycolysis? As a means to study this question we will develop methods to isolate and culture pure populations of the erythroid precursor, CFU-E, and pure populations of very young circulating reticulocytes from rats. The age and homogeneity of these cells will be determined by the extent and variability of binding fluourescein-labelled transferrin; a marker for erythroid cell age. In this way, cell samples from the entire developmental period from CFU-E to mature red cells can be studied. The isozymes of PK present during erythroid development will be purified and monospecific antibodies will be produced. We will investigate synthesis, degradation and secondary modification as processes controlling the final activity of PK in mature red cells. Particular attention will be paid to the potentially exciting role of subcellular binding of PK as protective mechanism against degradation. The role of ubiquitin and ATP in PK degradation will be assessed. Secondary modification of PK will be examined by charting the appearance of the proteolytically cleaved PK-R' subunit. The role of phosphorylation of PK in appearance of PK-R' will be investigated. Finally, we will determine which developmental process is controlled by the Dpg gene in rats. Animals of different genotype at this locus are mutant with respect to one of the developmental processes we will investigate since mature red cells differ by 40% in activity of two rate-limiting glycolytic enzymes: PK and phosphofructokinase. The proposed studies will greatly advance knowledge of processes controlling the level of one key factor in mature red cell metabolism: PK activity, and will pinpoint the developmental process affected by Dpg.